Rat Aβ1-40 ELISA Kit
Rat Aβ1-40 ELISA Kit
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| Usage | Sample Processing and Requirements: Serum: Place whole blood specimens collected in serum separator tubes at room temperature for 2 hours or at 4°C overnight, then centrifuge at 1000×g for 20 minutes. Remove the supernatant for testing, or store the supernatant at -20°C or -80°C. Avoid repeated freezing and thawing. Plasma: Collect specimens using EDTA or heparin as an anticoagulant. Centrifuge at 1000×g for 15 minutes at 2-8°C within 30 minutes of collection. Remove the supernatant for testing, or store the supernatant at -20°C or -80°C. Avoid repeated freezing and thawing.Other biological fluids: Centrifuge at 1000xg for 20 minutes, remove the supernatant and test. Pre-test preparation: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Preparation of standard gradient working solution: Add 1mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, then gently mix (concentration is 1000pg/mL). Then dilute to the following concentrations: 1000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, 15.625pg/mL, and 0pg/mL. Serial dilution method: Take 7 EP tubes and add 500μL of universal diluent to each tube. Pipette 500μL of the 1000pg/mL standard working solution into the first EP tube and mix thoroughly to make a 500pg/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube is directly used as a blank well; there is no need to pipette liquid from the penultimate tube. 3. Preparation of biotinylated detection antibody working solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration with universal diluent (e.g., 10μL concentrate + 990μL universal diluent). Prepare immediately before use. 4. Prepare the enzyme conjugate working solution: 15 minutes before use, centrifuge the 100× concentrated enzyme conjugate at 1000×g for 1 minute. Dilute the 100× concentrated HRP enzyme conjugate to a 1× working concentration with universal diluent (e.g., 10 μL of concentrate + 990 μL of universal diluent). Prepare freshly for use. 5. Prepare the 1× wash solution: Dispense 10 mL of 20× wash solution into 190 mL of distilled water. (Concentrated wash solution removed from the refrigerator may crystallize; this is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing.) Procedure: 1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes. Seal the remaining strips in a ziplock bag and return them to 4°C. 2. Sample addition: Add 100 μL of sample or standard of varying concentrations to the corresponding wells. Add 100 μL of universal diluent to the blank wells. Cover with film and incubate at 37°C for 60 minutes. (Recommendation: Dilute the sample to be tested at least 1-fold with universal diluent before adding it to the ELISA plate for testing. This will minimize the impact of matrix effects on the test results. The final calculation of sample concentration should be multiplied by the corresponding dilution factor. It is recommended to run duplicate wells for all test samples and standards.) 3. Add Biotinylated Antibody: Remove the ELISA plate and discard the liquid. Do not wash. Add 100 μL of Biotinylated Antibody Working Solution directly to each well. Cover with a film sealer and incubate at 37°C for 60 minutes. 4. Wash: Discard the liquid. Add 300 μL of 1x Wash Solution to each well. Let stand for 1 minute. Shake off the wash solution and pat dry on absorbent paper. Repeat this process three times (a plate washer can also be used). 5. Add Enzyme Conjugate Working Solution: Add 100 μL of Enzyme Conjugate Working Solution to each well. Cover with a film sealer and incubate at 37°C for 30 minutes. 6. Wash: Discard the liquid and wash the plate five times according to the washing method in step 4. 7. Add substrate: Add 90uL of substrate (TMB) to each well, cover with sealing film, and incubate at 37℃ in the dark for 15 minutes. 8. Add stop solution: Remove the ELISA plate and add 50uL of stop solution directly to each well. Immediately measure the OD value of each well at a wavelength of 450nm. Calculation of experimental results: Result evaluation: 1. Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as the correction value. With concentration as the horizontal axis and OD value as the vertical axis, draw a standard curve of the four-parameter logistic function on double logarithmic coordinate paper. 2. If the sample OD value is higher than the upper limit of the standard curve, it should be diluted appropriately and re-measured and multiplied by the corresponding dilution factor when calculating the sample concentration. |
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| Theory | This kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with an amyloid beta peptide 1-40 (Aβ1-40) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of amyloid beta peptide 1-40 (Aβ1-40) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||
| Synonym | Rat Amyloid Beta Peptide 1-40 ELISA Kit | |||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||
| Composition |
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| Background | Amyloid-β (Aβ) is a 39-43 amino acid polypeptide produced by the proteolytic hydrolysis of amyloid precursor protein by β- and γ-secretases. It is produced by a variety of cells and circulates in the blood, cerebrospinal fluid, and interstitial fluid, mostly bound to chaperone protein molecules, with a small amount existing in a free state. The most common Aβ isoforms are Aβ1-40 and Aβ1-42. Aβ1-40 is present at levels 10-fold and 1.5-fold higher than Aβ1-42 in CSF and blood, respectively. Aβ1-40 is produced in the TGN and its secretory vesicles, representing the secretory form of Aβ. | |||||||||||||||||||||||||||||||
| General Notes | 1. Strictly adhere to the specified incubation time and temperature to ensure accurate results. All reagents must be at room temperature (20-25°C) before use. Refrigerate reagents immediately after use. 2. Improper plate washing may lead to inaccurate results. Ensure that the wells are completely aspirated before adding substrate. Do not allow the wells to dry out during incubation. 3. Remove any residual liquid and fingerprints from the bottom of the plate, as this will affect the OD value. 4. The substrate developer solution should be colorless or very light in color. Do not use substrate solution that has turned blue. 5. Avoid cross-contamination of reagents and specimens to prevent erroneous results. 6. Avoid direct exposure to strong light during storage and incubation. 7. Do not expose any reagents to bleaching solvents or the strong fumes emitted by bleaching solvents. Any bleaching component will destroy the biological activity of the reagents in the kit. 8. Do not use expired products, and do not mix components from different product numbers and batches. 9. Recombinant proteins from sources other than the test kit may not match the antibodies in this kit and may not be recognized. 10. If there is a possibility of disease transmission, all samples should be managed properly and the samples and detection devices should be handled according to the prescribed procedures. |
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| Storage Temp. | Store at 2-8°C. Valid for 6 months. | |||||||||||||||||||||||||||||||
| Test Range | 15.62-1000 pg/mL | |||||||||||||||||||||||||||||||
| Applications | Serum, plasma, and other biological fluids |
